Expression of Intimin g from Enterohemorrhagic Escherichia ... · The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule intimin include the recep-tor-binding

INFECTION AND IMMUNITY,0019-9567/00/$04.0010

Aug. 2000, p. 4637–4646 Vol. 68, No. 8

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Expression of Intimin g from EnterohemorrhagicEscherichia coli in Citrobacter rodentium

ELIZABETH L. HARTLAND,1 VERONIKA HUTER,1† LISA M. HIGGINS,2 NATHALIE S. GONCALVES,2

GORDON DOUGAN,1 ALAN D. PHILLIPS,3 THOMAS T. MACDONALD,2 AND GAD FRANKEL1*

Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AZ,1

Department of Paediatric Gastroenterology, St. Bartholomew’s and the Royal London Schoolof Medicine and Dentistry, St. Bartholomew’s Hospital, London EC1A 7BE,2 and

University Department of Paediatric Gastroenterology, Royal Free Hospital,London NW3 2QG,3 United Kingdom

Received 10 February 2000/Returned for modification 18 April 2000/Accepted 17 May 2000

The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule intimin include the recep-tor-binding domain. At least five different types of Int280, designated a, b, g, d, and «, have been describedbased on sequence variation in this region. Importantly, the intimin types are associated with different evolu-tionary branches and contribute to distinct tissue tropism of intimin-positive bacterial pathogens. In this studywe engineered a strain of Citrobacter rodentium, which normally displays intimin b, to express intimin g fromenterohemorrhagic Escherichia coli. We show that intimin g binds to the translocated intimin receptor (Tir)from C. rodentium and has the ability to produce attaching and effacing lesions on HEp-2 cells. However, C. ro-dentium expressing intimin g could not colonize orally infected mice or induce mouse colonic hyperplasia.These results suggest that intimin may contribute to host specificity, possibly through its interaction with areceptor on the host cell surface.

Enteropathogenic Escherichia coli (EPEC) is an importantcause of severe infantile diarrheal disease in many parts of thedeveloping world. EPEC bacteria colonize the small intestinalmucosa and, by subverting intestinal epithelial cell function,produce a characteristic histopathological feature known asthe “attaching and effacing” (A/E) lesion (11). The A/E lesionis characterized by localized destruction (effacement) of brushborder microvilli, intimate attachment of the bacillus to thehost cell membrane, and the formation of an underlying ped-estal-like structure in the host cell. Similar lesions have beenassociated with several other bacterial mucosal pathogens, in-cluding enterohemorrhagic E. coli (EHEC) (23) and Citro-bacter rodentium (28). EHEC is a food-borne pathogen ofworldwide importance which can cause acute gastroenteritis,hemorrhagic colitis, and hemolytic uremic syndrome (23). C. ro-dentium is the causative agent of transmissible murine colonichyperplasia (3), a disease of laboratory mice characterized bycrypt hyperplasia, epithelial cell proliferation, crypt dilation,mucosal thickening, and the development of an uneven epi-thelial surface of the descending colon.

The first gene to be associated with A/E activity was eae,which encodes the bacterial adhesion molecule intimin (16).Mutational analysis of the eae genes of EPEC, EHEC, andC. rodentium has shown that intimin is necessary for coloniza-tion of the host and disease (7, 16, 29). Using human intestinalorgan cultures as an infection model system, intimin has beenshown to be essential for colonization of the mucosa and A/Elesion formation (13).

Recently, we and others described five distinct intimin sub-types, intimin a, b, g, d, and ε, based on sequence variation

within the C-terminal 280-amino-acid receptor-binding do-main of the polypeptide (Int280) (1, 25). Intimin a is specifi-cally expressed by a group of EPEC strains, all of which belongto one evolutionary branch of EPEC known as EPEC clone 1(32), and Hafnia alvei. Intimin b is mainly associated withEPEC and EHEC strains belonging to their respective clones2, C. rodentium and rabbit diarrheagenic E. coli type 1, whileintimin g is associated with EHEC O157:H7 and EPEC O55:H7 (1). This observation raises the possibility that tissue tro-pism exhibited by EPEC (colonizing mainly the small bowel)and EHEC O157 (colonizing the large bowel) is intimin re-lated. Intimin exchange studies have been performed in pigletsusing wild-type EHEC (expressing intimin g) or EHEC ex-pressing the EPEC-derived intimin a. In conventional animals,no differences were seen in intestinal distribution of the A/Elesions (7). However, in gnotobiotic piglets, EHEC expressingintimin a produced A/E lesions in both the small and largeintestines whereas EHEC expressing intimin g produced le-sions only in the large intestine (30). More recently, we haveshown, using human intestinal explants, that EHEC O157:H7expressing intimin g can colonize and induce A/E lesion onlyon the follicle-associated epithelium of the Peyer’s patch(27). In contrast, EPEC O127:H6 expressing intimin a (strainE2348/69) colonized Peyer’s patch as well as proximal anddistal small intestinal tissues (27). Importantly, tissue tropismtowards Peyer’s patches was observed following expression ofintimin g in the EPEC background (26). These results suggestthat different intimins may play a role in determining the pat-tern of colonization and tissue tropism in the host.

We and others have shown that Int280 (from EPEC, EHEC,and C. rodentium) can bind directly to uninfected host cells (2,8). However, intimin can also bind to the bacterial receptor,Tir (EspE), which is translocated by the bacteria into the hostcell membrane during infection (5, 18). Recently, the globalfold of Int280a in solution was determined by multidimen-sional nuclear magnetic resonance (17). The structure showsthat Int280 comprises three separate domains, two immuno-

* Corresponding author. Mailing address: Department of Biochem-istry, Imperial College, London SW7 2AZ, United Kingdom. Phone:44-20-7594-5253. Fax: 44-20-7594-5255. E-mail: [email protected].

† Present address: Institute of Microbiology and Genetics, Univer-sity of Vienna, A-1030 Vienna, Austria.

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globulin-like domains and a C-type lectin-like module. Mod-elling of other intimin types shows that these proteins possesssimilar structures, which define a new family of bacterial ad-hesion molecules.

Oral infection of mice with live wild-type C. rodentium (15)or intracolonic inoculation of dead bacteria (14) induces aCD31 and CD41 T-cell infiltration into the colonic laminapropria and a T-helper type 1 immune response. This re-sponse is not observed, however, in mice inoculated withbacteria lacking intimin, but is seen in mice inoculated withC. rodentium complemented with intimin a from EPEC en-coded on pCVD438 (14, 15). In this study, we replaced thereceptor-binding domain of intimin a on pCVD438 with itsintimin g homologue (generating plasmid pICC55). This hy-brid intimin was expressed in eae mutant strains of EPEC(CVD206) (6) and C. rodentium (DBS255) (28). Althoughbiologically functional, this form of intimin g could not restoremouse virulence when expressed in DBS255. These resultssuggest that the variable receptor-binding domain of intiminmay contribute to the species specificity exhibited by C. roden-tium and by other A/E bacterial pathogens.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains used in this study wereE. coli BL21 and TG1 and wild-type C. rodentium, EPEC (strain E2348/69), andtheir eae deletion mutants, strains DBS255 (28) and CVD206 (6), respectively.The plasmids used in this study are listed in Table 1. Plasmid pCVD438 is apACYC184 vector harboring the intimin a-encoding eae gene from EPECE2348/69 (6). pCVD444 is a pUC18 vector harboring the intimin g-encoding eaegene from EHEC EDL933 (33). Bacteria were grown at 37°C in L broth orDulbecco’s modified Eagle’s medium. Where appropriate, chloramphenicol andampicillin were added to final concentrations of 30 and 100 mg/ml, respectively.

Construction of hybrid intimin g and maltose-binding protein (MBP)-Int280gfusion protein. A schematic representation of the strategy used to produce thehybrid intimin g is shown in Fig. 1. In order to replace the receptor-bindingdomain of intimin a in pCVD438 with the intimin g homologue, we took ad-vantage of two unique restriction endonuclease sites located in pCVD438, aconserved SalI site located upstream of the receptor binding domain (position1663 of the eae gene) and an EagI site located downstream to the TAA stopcodon and within the pACYC184 vector plasmid (9). The DNA fragment be-tween the SalI site and the 39 end of the eae gene encoding intimin g frompCVD444 was amplified by PCR using a forward primer (1606-59 GGC AATAGC TCT AAC AAT GTA) and an eae g-derived reverse primer overlappingthe 39 end of the gene and including an EagI restriction endonuclease site (59CTT ACA TGR AGC ATC AGC ATA ATA GGC TTG), as previously de-scribed (8). The amplified eae fragment, flanked by SalI and EagI restrictionsites, was used to replace the corresponding fragments of pCVD438 as previouslydescribed (9) (Fig. 1). Following confirmation by DNA sequencing, the modifiedplasmid, pICC55 (Table 1), was transformed into the eae deletion mutants ofEPEC and C. rodentium, strains CVD206 and DBS255, respectively, by electro-poration.

The DNA fragment encoding Int280 from pICC55 was amplified by PCR(forward, 59-GGAATTCATTACTGAGATTAAGGCT-39; reverse, 59-CCCAAGCTTTTATTYTACACAA-39) and subcloned into pMALc-2, producing plas-mid pICC58, in E. coli TG1, as previously described (8). Expression of MBP-Int280g was induced by IPTG (isopropyl-b-D-thiogalactopyranoside), and thefusion protein was purified as previously described (8).

Detection of intimin expression by Western blotting and FAS. Expression ofthe intimin derivatives was determined by Western blotting (4). Briefly, station-ary L broth cultures were diluted 1:100 in Dulbecco’s modified Eagle’s mediumand incubated for 3 h at 37°C. An equivalent of a culture at an optical density at600 nm (OD600) of 0.5 was loaded onto sodium dodecyl sulfate–7.5% polyacryl-amide gel electrophoresis (SDS–7.5% PAGE) as described (4). The electropho-resed polypeptides were transferred to a nitrocellulose membrane, and immu-nodetection of intimin was performed using a universal intimin antiserum raisedin rabbits against a conserved intimin fragment (amino acids Gly388 to Lys667)(4) diluted 1:500. Fluorescence actin staining (FAS) test was employed for 3 and6 h to detect A/E lesion formation on HEp-2 cells as described previously (20,24).

Preparation of C. rodentium His-Tir and gel overlays. The DNA segmentencoding Tir in C. rodentium (Tir-Cr) was amplified using C. rodentium DNAas template (primers: forward, 59-GAAGATCTATGCCTATTGGTAATCTTG

FIG. 1. Schematic representation showing the construction of recombinantintimin g. The N-terminal and receptor binding regions of intimin a and intiming are represented by different shades. The percentage amino acid identity of eachregion of intimin a and intimin g is given in brackets. The position of the re-striction site, SalI, used to construct the recombinant intimin is given in nucle-otides, and the position corresponding to SalI in intimin is represented as adashed line. Schematic is not to scale.

FIG. 2. Western blot analysis of C. rodentium strains expressing intimin a andthe hybrid intimin g. Whole-cell protein preparations were separated by SDS-PAGE, and intimins were detected with polyclonal rabbit antibodies raised to aconserved region of intimin. Lane 1, DBS255; lane 2, DBS255(pCVD438); lane3, DBS255(pICC55).

TABLE 1. Plasmids used in this study

Plasmid Property Reference or source

pCVD438 pACYC184 encoding intimin a 6pCVD444 pUC18 encoding intimin g 33pET28a Vector for expression of His-tagged

proteinsNovagen

pICC55 pCVD438 derivative encodingrecombinant intimin g

This study

pICC56 pMALc-2 expressing MBP-Int280g This studypICC58 pET28a expressing His-Tir-Cr This studypMALc-2 Vector for generating MBP fusions New England Biolabs

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GT; reverse, 59-GAAGATCTTTAGACGAAACGATGGGATC). The tir frag-ment was sequenced and cloned into the BamHI site of pET28a (generatingplasmid pICC56) in E. coli BL21 previously described (12). For gel overlays,whole-bacterial-cell protein extract from E. coli BL21a(pICC56) expressingTir-Cr was separated by SDS-PAGE, blotted onto a nitrocellulose membrane asdescribed above, and blocked with 10% skim milk in phosphate-buffered saline(PBS)–0.1% Tween 20 overnight. The nitrocellulose membranes were reactedwith either rabbit anti-Tir antibodies (12) or with 5 mg of the purified MBP-Int280 fusion proteins per ml in PBS–0.1% Tween 20 for 2 h and washed twicefor 5 min in PBS–0.1% Tween 20. Binding of MBP fusions or Tir antibodies toTir-Cr was detected with rabbit anti-MBP antiserum (1:2,000 for 1 h) and/oranti-rabbit antibodies conjugated to alkaline phosphatase (1:2,000 for 1 h) (12).

IVOC adhesion assay. Tissue was obtained, with fully informed parental con-sent and ethics approval, using grasp biopsy forceps during routine endoscopic(Olympus PCF pediatric endoscope) investigation of intestinal disorders. Ter-minal ileal Peyer’s patch tissue was taken from patients from areas showing noendoscopic abnormality. IVOC infection was performed as described previously(13). The assay was terminated at 8 h. Each bacterial strain was examined in in

vitro organ cultures (IVOC) on least three occasions by using tissue from dif-ferent children.

Challenge of mice with C. rodentium. Mice (Swiss NIH, C3H) were orallyinoculated by gavage with C. rodentium, DBS255(pCVD438), DBS255(pICC55),and DBS255. Bacteria were diluted with PBS (pH 7.2) to an OD600 of 1.7 anddelivered to mice in a volume of 100 ml as previously described (15). Mice werekilled at 12 days postchallenge, and tissue was snap frozen in liquid nitrogen andstored at 270°C for further analysis.

Immunohistochemistry. Three-step avidin-peroxidase staining was performedon 5-mm-diameter frozen sections as described previously (15) using monoclonalantibodies 145-2C11 (anti-CD3) and YTS 191 (anti-CD4). Biotin-conjugatedrabbit anti-rat immunoglobulin G (IgG) (DAKO, High Wycombe, United King-dom) and goat anti-hamster IgG (Vector Laboratories, Peterborough, UnitedKingdom) were used at a 1:50 dilution in Tris-buffered saline (TBS) (pH 7.6)containing 4% (vol/vol) normal mouse serum (Harlan Seralab, Oxon, UnitedKingdom). Avidin peroxidase (Sigma) was used at a dilution of 1:200 in TBS. Atwo-step protocol was performed using rabbit anti-intimin antibody (4) togetherwith horseradish peroxidase-conjugated swine anti-rabbit IgG secondary anti-

FIG. 3. Infection of healthy human tissue obtained from terminal ileal Peyer’s patch of children with CVD206 carrying various plasmids. (A) CVD206(pCVD438).(B) CVD206(pICC55).

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body. Peroxidase activity was detected with 3,39-diaminobenzidine-tetra-hydro-chloride (DAB; Sigma) in 0.5 mg of Tris-HCl (pH 7.6) per ml containing 0.01%H2O2. Endogenous-peroxidase-containing cells were visualized by incubation ofsections with DAB substrate and H2O2 alone. The density of positive cells in thelamina propria was determined by image analysis as described previously (15).

Inoculation of dead bacteria and analysis of hyperplasia and cellular infil-trate. Formalin-killed bacteria (5 3 108) were injected into the colon of BALB/cmice treated 15 min previously with 0.1 ml of 50% ethanol to break the mucosalbarrier (14). Six days later, mice were killed and the colon was dissected out. Theweight of the terminal 4 cm of distal colon was determined, and then the samplewas immediately snap frozen in liquid nitrogen for immunohistochemistry. Mu-cosal thickness (i.e., crypt length) was measured on well-oriented sections ofcolon by using a calibrated eyepiece graticule as described previously (14). Atleast five measurements were made per sample, and there were five mice pergroup. The densities of CD3, CD4, and CD81 cells in the lamina propria wereanalyzed using a Seescan image analyzer as previously described (15).

RESULTS

Construction of hybrid intimin g. We have shown previouslythat wild-type C. rodentium (expressing intimin b) and C. ro-dentium expressing the EPEC-derived intimin-a [DBS255(pCVD438)] colonize and produce A/E lesions only in thedescending colon of orally challenged mice (10). In order toinvestigate the tissue selectivity exhibited by C. rodentiumexpressing intimin g, we expressed this intimin in DBS255 fromthe high-copy-number, pUC18-derived plasmid pCVD444 (33).A cat cassette (pCVD444*) was inserted into this vector toovercome the natural ampicillin resistance of C. rodentium(data not shown). Only low levels of intimin were detected inwhole-cell extracts of DBS255(pCVD444*) (data not shown).In order to circumvent poor expression, we replaced the re-ceptor-binding domain of intimin a on pCVD438, which wasalready shown to mediate high levels of intimin expression onthe cell surface of DBS255, with that of intimin g (see Mate-rials and Methods and Fig. 1) to produce a plasmid, pICC55.Since the amino-terminal 554 amino acids of intimin a en-coded by the recombinant eae gene on pICC55 are 97% iden-tical to the homologous region of intimin g (Fig. 1), the hybridintimin is, for all intents and purposes, intimin g. The hybridintimin g was expressed in DBS255(pICC55) and CVD206(pICC55), and its biological activity was tested. The isogenicstrains DBS255(pCVD438) and CVD206(pCVD438) were usedas controls.

Expression of intimin was determined by Western blot anal-ysis of whole-cell lysates prepared from the different DBS255and CVD206 derivatives by using a universal, broad-spectrumpolyclonal intimin antiserum reactive with all the different in-timin types (4). Lysates from all the recombinant strains, butnot from CVD206 or BDS255, reacted with the antiserum (Fig.2 and data not shown), indicating that the hybrid intimin g isexpressed in DBS255 at a level comparable to that of intimin aand to those of the recombinant CVD206 strains.

Interaction of CVD206 expressing the hybrid intimin g withHEp-2 cells and human intestinal IVOC. Before the recombi-nant DBS255 strain was tested in the mouse model, the hy-brid intimin g was subjected to a number of in vitro biologicalassays, using CVD206(pICC55), designed to determine the in-fluence of the genetic manipulation on the function of thisconstruct. Firstly, the ability of the hybrid intimin g to me-diate A/E lesion formation on HEp-2 cells was investigated.CVD206(pICC55) adhered to cell monolayers in a localizedpattern and produced a FAS-positive reaction, similar to thatof CVD206(pCVD438) (data not shown). The result showsthat this intimin g can mediate A/E lesion formation on cul-tured human epithelial cells.

In previous studies, we have shown that the ability of EPECto induce A/E lesions on human intestinal IVOC is dependenton surface expression of biologically active intimin (13). To

test the ability of the hybrid intimin g to mediate A/E lesionformation on mucosal surfaces, healthy human tissue ob-tained from terminal ileal Peyer’s patch of children was ex-amined after infection with CVD206(pICC55). Like CVD206(pCVD438), CVD206(pICC55) was able to form A/E lesionson the human tissue (three out of three incubations) (Fig. 3).No binding was observed with CVD206 only (data not shownand reference 12). These results show that pICC55 encodes abiologically functional intimin that can mediate binding and A/E lesion on mucosal surfaces.

Colonization of the mouse intestine following live oral in-fections with C. rodentium. Following verification of the bind-ing activity of the hybrid intimin g in EPEC, we confirmed itsability to restore A/E lesion formation on HEp-2 cells toDBS255 and tested mouse virulence of DBS255(pICC55). Totest the ability of DBS255(pICC55) to mediate A/E lesionformation on HEp-2 cells, monolayers were infected withDBS255(pCVD438) or DBS255(pICC55) cells for 6 h as pre-viously described (24). Both DBS255(pCVD438) and DBS255(pICC55) cells adhered poorly to HEp-2 cell monolayers, butFAS-positive bacteria were observed for both strains (Fig. 4).

For virulence assays, mice were killed on day 12 postinfectionand colons were examined for signs of bacterial colonization andmucosal hyperplasia by immunohistochemistry. The presence ofbacteria adhering to the epithelial surface was visualized by im-munohistochemistry using the intimin antibody (4) (Fig. 5).Citrobacter bacteria were detected in mice infected with thewild-type C. rodentium and DBS255(pCVD438) strains, whileno bacteria could be seen in mice infected with DBS255 orDBS255(pICC55) (Fig. 5). When bacteria were present, they

FIG. 4. Fluorescent actin staining of HEp-2 cell monolayers infected withDBS255(pCVD438) (A) and DBS255(pICC55) (B). Arrows indicate FAS-posi-tive bacteria.



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were observed in close association with enterocytes. However,although adhesion remained tissue specific, the cell specificityof DBS255(pCVD438) appeared to be altered, as there was amore extensive colonization of the crypts, confirming our pre-vious observations (10). Colonization of the epithelium wasdirectly associated with mucosal thickening and crypt hyper-plasia. Indeed, the colons from wild-type C. rodentium- andDBS255(pCVD438)-infected mice were very similar. Macro-scopic thickening of the distal colon was observed in all mice inthese groups. Microscopic examination showed massive epi-thelial cell hyperplasia and CD4 cell infiltrate with an increasein mucosal thickness and crypt length (Fig. 6A and B). Thecolons of mice challenged with DBS255, DBS255(pICC55),or DBS255(pCVD444*) did not show any histopathologicalchanges, and the tissue was indistinguishable from those ofuninfected control mice (Fig. 6C and D).

The hybrid intimin g binds Tir from C. rodentium. The factthat the hybrid intimin g confers A/E lesion formation activityto DBS255(pICC55) on HEp-2 cells indicates that it binds Tirfrom C. rodentium (Tir-Cr). In order to confirm this exper-imentally, gel overlay binding assays were used to probe forthe interaction of the hybrid intimin g with Tir-Cr. For thispurpose, the DNA fragment encoding Tir-Cr was amplified by

PCR and cloned into the expression vector pET28a (generatingplasmid pICC56) in E. coli BL21. Partial sequencing of the N-and C-terminal regions of Tir-Cr revealed exact homology witha Tir sequence derived from mouse enteropathogenic E. coli(MPEC) (accession no. AB 026719) (22). In parallel, the DNAfragment encoding the carboxy-terminal 280-amino-acid re-ceptor-binding domain of the hybrid intimin g (Int280g) wasamplified by PCR and cloned into pMAL-c vector (gener-ating plasmid pICC58) in E. coli TG1. Int280g was overex-pressed and purified as an MBP fusion (MBP-Int280g). MBP-Int280a and MBP were used as positive and negative controls,respectively.

Expression of the recombinant His-Tir-Cr was induced byIPTG. Whole-cell extracts were then blotted, following elec-trophoresis, onto nitrocellulose membranes, and the immobi-lized Tir-Cr was allowed to react with either the MBP-Int280aor MBP-Int280g fusion proteins or MBP or with anti-Tir an-tiserum (12). Binding of the intimin derivatives was detectedfollowing further incubations with rabbit anti-MBP and/or al-kaline phosphatase-conjugated anti-rabbit antisera. Theseoverlay experiments, although they did not provide a quanti-tative measure, showed that similarly to anti-Tir antibodies,

FIG. 5. Intimin staining of C. rodentium-infected colonic tissue. DBS255(pCVD438) expressing intimin a is seen colonizing deep down into crypts (A) and wild-typeC. rodentium expressing intimin b is seen adhering to surface exposed epithelium (B), while no bacteria can be seen following infection with DBS255 cells (C) or DBS255(pICC55) cells expressing the hybrid intimin g (D).



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both Int280a and the hybrid Int280g could bind Tir-Cr (Fig. 7).No binding was detected using MBP only (data not shown).

Mouse colonic hyperplasia and T-cell infiltration. In a pre-vious study, we showed that intracolonic inoculation of micewith dead C. rodentium expressing either intimin b or intimina induced T-cell infiltration to the lamina propria at the baseof the crypts and colonic inflammation and hyperplasia withoutdetectable bacterial colonization. To test for the Tir-indepen-

dent biological function of the hybrid intimin g in vivo, weinvestigated T-cell infiltration of the colonic submucosa fol-lowing inoculation with dead DBS255(pICC55) bacteria. Incontrast to inoculation with wild-type and intimin a-expressingC. rodentium, no T-cell infiltration or increases in colonicweight or mucosal thickness were detected following infectionwith C. rodentium expressing the hybrid intimin g or DBS255(Fig. 8). Taken together, these results show that the hybrid

FIG. 6. Mucosal thickness in mice 12 days after inoculation with live wild-type C. rodentium (A), DBS255(pCVD438) (B), DBS255 (C), and DBS255(pICC55) (D).Note that in panels A and B, the mucosa is thick and hyperplastic with an increase in CD41 cells (arrows), in contrast to panels C and D, in which the colon is thinand essentially normal and there are very few CD41 cells (immunoperoxidase with anti-CD4; magnification, 3200).



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intimin g cannot restore mouse virulence to C. rodentium and,unlike intimin a and b, it has no biological activity in themurine situation.

DISCUSSION

Intimin was the first gene product of EPEC to be associatedwith A/E lesion formation (16). Recently, the eae genes fromdifferent A/E lesion forming bacterial pathogens were catego-rized into a family of antigenically distinct intimin types (a, b,

g, d, ε) (1, 25). Studies on the different intimins from EPEC,EHEC, and C. rodentium have shown that receptor-bindingactivity is localized to the C-terminal 280 amino acids (Int280)(8). A number of groups have reported that intimin can binddirectly to uninfected host cells (2, 8) and to a receptor en-coded by the bacteria, termed Tir, which is translocated intothe host cell membrane via a type III secretion system (18).Binding to the host cell but not to Tir is dependent on a di-sulfide bridge at the carboxy terminus of Int280 (12). However,when expressed on the surface of EPEC, both of these binding

FIG. 6—Continued.



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activities of intimin are required for intimate bacterial adhe-sion and A/E lesion formation.

Recently, the global fold of Int280a in solution was deter-mined by multidimensional nuclear magnetic resonance (17).The structure shows that Int280 is built from three globulardomains: D1 (residues 1 to 91), D2 (residues 93 to 181), andD3 (residues 183 to 280). The first two domains, D1 and D2,although lacking disulfide bonds, resemble the type I set of theIg super family (IgSF). The IgSF domains in intimin appear toform an articulated linker that most likely extends away fromthe bacterium surface and confers a highly accessible thirddomain, D3 (residues 183 to 280), for potential interaction.Despite the lack of significant sequence homology, the topol-ogy in Int280 D3 is reminiscent of C-type lectins, a family ofproteins responsible for cell surface carbohydrate recognition.These findings imply that carbohydrate recognition may beimportant for intimin-mediated cell adhesion, which in turnmay provide a mechanism for tissue tropism exhibited by dif-ferent A/E lesion forming bacterial pathogens. Indeed, a recentstudy by Vanmaele et al. (31) showed that coincubation ofEPEC with Lewis X-bovine serum albumin glycoconjugatecaused a decrease in intimin expression by the bacteria. Theseresults are consistent with our previous observations of downregulation of intimin expression following A/E lesion forma-tion (19).

Intimin exchange studies performed in piglets suggest thatdifferent intimin types might determine tissue tropism. In thesestudies, wild-type EHEC bacteria (expressing intimin g) orEHEC bacteria expressing the EPEC-derived intimin a wereused. In conventional animals (7), no differences were seen inthe intestinal distribution of the A/E lesions, but in gnotobioticpiglets (30), EHEC expressing intimin a produced A/E lesionsin both the small and large intestines whereas EHEC express-ing intimin g produced lesions only in the large intestine. Morerecently we have shown that intimin contributes to tissue tro-pism exhibited by EPEC (colonizing all regions of human smallintestinal explants) and EHEC (which specifically target thefollicle-associated epithelium of the Peyer’s patch) (26, 27).The aim of the present study was to extend these investigationsfurther to study the contribution of the intimin types to host(species) specificity.

A difficulty associated with working on EPEC and EHEC isthe lack of a small animal model for studying the biologicalproperties of EPEC-associated genes in an in vivo situation.C. rodentium causes transmissible colonic hyperplasia in mice

(3), an infection associated with the formation of A/E lesionssimilar to those described for human EPEC (28). C. rodentiumhas been shown to harbor the loss of enterocyte effacementpathogenicity island encoding an eae homologue (21) that di-rects the expression of an intimin b protein that is essential forA/E lesion formation and infection of mice (29). Moreover,expression of intimin a from EPEC restores the ability ofDBS255 to colonize the colon of orally challenged mice (10).This model provides an opportunity to evaluate the in vivobiological functions of different intimin types in mice.

In the present study, we determined the outcome of mouseinoculation with DBS255 bacteria expressing intimin g. Sincelow intimin expression was observed in DBS255 bacteria ex-pressing intimin g from a pUC18-cloned eae gene (pCVD444*),we generated a hybrid intimin g based on pCVD438 expressingintimin a. This intimin contains the receptor-binding domain

FIG. 7. Gel overlays showing binding of MBP-Int280 fusions to Tir from C.rodentium (Tir-Cr). Tir-Cr was expressed as a His-tagged fusion. Induced whole-cell protein preparations were separated by SDS-PAGE and overlaid with dif-ferent purified MBP-Int280’s. Lanes 1, 3, and 5: E. coli BL21 carrying pICC56;lanes 2, 4, and 6: E. coli BL21 carrying pET28a alone (included as a negativecontrol). Blots were overlaid with anti-Tir antiserum (lanes 1 and 2), MBP-Int280g(lanes 3 and 4), or MBP-Int280a (lanes 5 and 6).

FIG. 8. (A) Weights of the distal colons of mice. (B) Crypt lengths are shown;only the DBS255(pCDV438) strain intimin produced hyperplastic crypts. (C)Numbers of CD31, CD41, and CD81 cells in the lamina propria. In panels Athrough C, all mice were given 50% ethanol followed by DBS255(pCVD438)(white bars), the DBS255 strain (black bars), or DBS255(pICC55) (hatched bars)(n 5 5; p, P , 0.05, Mann-Whitney U test with Bonferroni’s correction). All dataare shown as the mean 6 1 standard error of the mean.



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of intimin g, presented on a cloned intimin a backbone whichitself is 97% identical to intimin g. This clone of intimin a hasalready been shown to mediate efficient intimin expression andto restore mouse virulence to DBS255.

Before using the recombinant eae gene in the C. rodentiummouse model, we confirmed that it encodes a biologically ac-tive intimin. This was achieved by expressing the hybrid intiming in CVD206(pICC55) bacteria. The hybrid intimin g was pro-duced in CVD206(pICC55) bacteria at a level similar to that ofintimin a in CVD206(pCVD438) bacteria. CVD206(pICC55)bacteria adhered and induced A/E lesions on HEp-2 cells.Importantly, this strain colonized and induced A/E lesionson human intestinal IVOC. Following these bioassays, weexpressed the hybrid intimin g in DBS255 cells. Using liveDBS255(pICC55) bacteria to infect HEp-2 cells, we showedthat the strain is capable of inducing A/E lesions and hence thehybrid intimin g is biologically functional in the C. rodentiumbackground and can cooperate with the other virulence factorsinvolved in this process. The ability of the hybrid intimin g tobind Tir-Cr was demonstrated using gel overlays and recom-binant proteins. In contrast, mice oral challenges revealed that,unlike intimin a, intimin g could not restore mouse virulenceto DBS255 bacteria. This suggests intimin g may exhibit a hostcell-encoded receptor binding specificity or affinity that differsfrom intimin a or intimin b and that this receptor is not ex-pressed, at least at a sufficient level, in the mouse gut. This re-sult is consistent with our data showing different tissue tropismbetween CVD206 expressing a and CVD206 expressing in-timin g, using IVOC from different regions of the human gut (26).

In previous studies, we have shown that following intraco-lonic inoculation of formalin-killed C. rodentium cells express-ing either intimin b or intimin a there was an extensive, Tir-independent, infiltration of CD31 CD41 T cells even thoughno bacteria were seen in association with the mucosa. For thisreason, we have examined the distal colon of mice inoculatedwith DBS255(pICC55) cells expressing the hybrid intimin g forsigns of T-cell infiltration. None of the mice inoculated withintimin g-encoding bacteria showed evidence of T-cell infiltra-tion or colonic hyperplasia. These data provide further evi-dence that intimin g does not bind to the mouse gut andsupports the role of a host cell intimin receptor in colonizationand disease. However, intimin is unlikely to be the only factorthat determines host specificity, as although C. rodentium ex-pressing intimin a causes colonic hyperplasia, as does intimina when presented to permeabilized rectum on dead EPECE2348/69 and CVD206(pCVD438) cells, these latter strainscannot colonize mouse colon or induce A/E lesions followinglive oral challenge. Accordingly, it seems that, like many othervirulence properties, host specificity is a multifactorial andmultigenic property of C. rodentium and EPEC.

ACKNOWLEDGMENTS

We thank Jim Kaper for providing bacterial strains and plasmidpCVD444, Michael Donnenberg for plasmid pCVD438, and DavidSchauer for DBS255. We thank Anton Page for his help with thephotography.

E.L.H. is the recipient of a Royal Society/NHMRC Howard FloreyFellowship. This work was supported by a grant from the BBSRC.

E.L.H. and V.H. contributed equally to this paper.

REFERENCES

1. Adu-Bobie, J., G. Frankel, C. Bain, A. G. Goncaleves, L. R. Trabulsi, G.Douce, S. Knutton, and G. Dougan. 1998. Detection of intimin a, b, g, andd, four intimin derivatives expressed by attaching and effacing microbialpathogens. J. Clin. Microbiol. 36:662–668.

2. An, H., J. M. Fairbrother, J. D. Dubreuil, and J. Harel. 1997. Cloning andcharacterization of the eae gene from a dog attaching and effacing Esche-

richia coli strain 4221. FEMS Microbiol. Lett. 148:239–245.3. Barthold, S. W., G. L. Coleman, R. O. Jacoby, E. M. Livstone, and A. M.

Jonas. 1976. Transmissiable murine colonic hyperplasia. Vet. Pathol. 15:223–236.

4. Batchelor, M., S. Knutton, A. Caprioli, V. Huter, M. Zanial, G. Dougan, andG. Frankel. 1999. Development of a universal intimin antiserum and PCRprimers. J. Clin. Microbiol. 37:3822–3827.

5. Deibel, C., S. Kramer, T. Chakraborty, and F. Ebel. 1998. EspE, a novelsecreted protein of attaching and effacing bacteria, is directly translocatedinto infected host cells, where it appears as a tyrosine-phosphorylated 90 kDaprotein. Mol. Microbiol. 28:463–474.

6. Donnenberg, M. S., and J. B. Kaper. 1991. Construction of an eae deletionmutant of enteropathogenic Escherichia coli by using a positive-selectionsuicide vector. Infect. Immun. 59:4310–4317.

7. Donnenberg, M. S., S. Tzipori, M. L. McKee, A. D. O’Brien, J. Alroy, andJ. B. Kaper. 1993. The role of the eae gene of enterohemorrhagic Escherichiacoli in intimate attachment in vitro and in a porcine model. J. Clin. Investig.92:1418–1424.

8. Frankel, G., D. C. A. Candy, P. Everest, and G. Dougan. 1994. Charac-terization of the C-terminal domains of intimin-like proteins of entero-pathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, andHafnia alvei. Infect. Immun. 62:1835–1842.

9. Frankel, G., A. D. Philips, M. Novakova, M. Batchelor, S. Hicks, and G.Dougan. 1998. Generation of Escherichia coli intimin-derivatives with differ-ing biological activities using site-directed mutagenesis of the intimin C-terminus domain. Mol. Microbiol. 29:559–570.

10. Frankel, G., A. D. Phillips, M. Novakova, H. Field, D. C. A. Candy, D. B.Schauer, G. Douce, and G. Dougan. 1996. Intimin from enteropathogenicEscherichia coli restores murine virulence to a Citrobacter rodentium eaeAmutant: induction of an immunoglobulin A response to intimin and EspB.Infect. Immun. 64:5315–5325.

11. Frankel, G., A. D. Phillips, I. Rosenshine, G. Dougan, J. B. Kaper, and S.Knutton. 1998. Enteropathogenic and enterohaemorrhagic Escherichia coli:more subversive elements. Mol. Microbiol. 30:911–921.

12. Hartland, E. L., M. Batchelor, R. M. Delahay, C. Hale, S. Matthews, G.Dougan, S. Knutton, I. Connerton, and G. Frankel. 1999. Binding of intiminfrom enteropathogenic Escherichia coli to Tir and to host cells. Mol. Micro-biol. 32:151–158.

13. Hicks, S., G. Frankel, J. B. Kaper, G. Dougan, and A. D. Phillips. 1998. Roleof intimin and bundle-forming pili in enteropathgenic Escherichia coli adhe-sion to pediatric intestinal tissue in vitro. Infect. Immun. 66:1570–1578.

14. Higgins, L. M., G. Frankel, I. Connerton, N. S. Goncalves, G. Dougan, andT. T. MacDonald. 1999. Role of bacterial intimin in colonic hyperplasia andinflammation. Science 285:588–591.

15. Higgins, L. M., G. Frankel, G. Douce, G. Dougan, and T. T. MacDonald.1999. Citrobacter rodentium infection in mice elicits a mucosal Th1 cytokineresponse and lesions similar to those in murine inflammatory bowel disease.Infect. Immun. 67:3031–3139.

16. Jerse, A. E., J. Yu, B. D. Tall, and J. B. Kaper. 1990. A genetic locus ofenteropathogenic Escherichia coli necessary for the production of attachingand effacing lesions on tissue culture cells. Proc. Natl. Acad. Sci. USA 87:7839–7843.

17. Kelly, G., S. Prasannan, S. Daniell, K. Fleming, G. Frankel, G. Dougan, I.Connerton, and S. Matthews. 1999. Structure of the cell-adhesion fragmentof intimin from enteropathogenic Escherichia coli. Nat. Struct. Biol. 6:313–318.

18. Kenny, B., R. DeVinney, M. Stein, D. J. Reinscheid, E. A. Frey, and B. B.Finlay. 1997. Enteropathogenic E. coli (EPEC) transfers its receptor forintimate adherence into mammalian cells. Cell 91:511–520.

19. Knutton, S., J. Adu-Bobie, C. Bain, A. D. Phillips, G. Dougan, and G.Frankel. 1997. Down regulation of intimin expression during attaching andeffacing enteropathogenic Escherichia coli adhesion. Infect. Immun. 65:1644–1652.

20. Knutton, S., T. Baldwin, P. H. Williams, and A. S. McNeish. 1989. Actinaccumulation at sites of bacterial adhesion to tissue culture cells: basis of anew diagnostic test for enteropathogenic and enterohemorrhagic Escherichiacoli. Infect. Immun. 57:1290–1298.

21. McDaniel, T. K., K. G. Jarvis, M. S. Donnenberg, and J. B. Kaper. 1995. Agenetic locus of enterocyte effacement conserved among diverse enterobac-terial pathogens. Proc. Natl. Acad. Sci. USA 92:1664–1668.

22. Nakagawa, M., R. Sakazaki, T. Muto, M. Saito, T. Hagiwara, and K.Imaizumi. 1996. Infectious megaenteron of mice. II. Detection of coliformorganisms of an unusual biotype as the primary cause. Jpn. J. Med. Sci. Biol.22:357–382.

23. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin.Microbiol. Rev. 11:142–201.

24. Newman, J. V., B. A. Zabel, S. S. Jha, and D. B. Schauer. 1999. Citrobacterrodentium espB is necessary for signal transduction and for infection oflaboratory mice. Infect. Immun. 67:6019–6025.

25. Oswald, E., H. Schmidt, S. Morabito, H. Karch, O. Marches, and A. Cap-rioli. 2000. Typing of intimin genes in human and animal enterohemorrhagic



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

and enteropathogenic Escherichia coli: characterization of a new intiminvariant. Infect. Immun. 68:64–71.

26. Phillips, A. D., and G. Frankel. 2000. Intimin-mediated tissue specificity inenteropathogenic Escherichia coli interaction with human intestinal organcultures. J. Infect. Dis. 181:1496–1500.

27. Phillips, A. D., S. Navabpor, S. Hicks, G. Dougan, T. Wallis, and G. Frankel.2000. Enterohaemorrhagic Escherichia coli O157:H7 targets Peyer’s patchesin man and causes attaching-effacing lesions in both human and bovineintestine. Gut, in press.

28. Schauer, D. B., and S. Falkow. 1993. Attaching and effacing locus of aCitrobacter freundii biotype that causes transmissible murine colonic hyper-plasia. Infect. Immun. 61:2486–2492.

29. Schauer, D. B., and S. Falkow. 1993. The eae gene of Citrobacter freundiibiotype 4280 is necessary for colonization in transmissible murine colonic

hyperplasia. Infect. Immun. 61:4654–4661.30. Tzipori, S., F. Gunzer, M. S. Donnenberg, L. de Montigny, J. B. Kaper, and

A. Donohue-Rolfe. 1995. The role of the eaeA gene in diarrhea and neuro-logical complications in a gnotobiotic piglet model of enterohemorrhagicEscherichia coli infection. Infect. Immun. 63:3621–3627.

31. Vanmaele, R. P., L. D. Heerze, and G. D. Armstrong. 1999. Role of lactosylglycan sequences in inhibiting enteropathogenic Escherichia coli attachment.Infect. Immun. 67:3302–3307.

32. Whittam, T. S., and E. A. McGraw. 1996. Clonal analysis of EPEC sero-groups. Rev. Microbiol. (Sao Paulo) 27(Suppl. 1):7–16.

33. Yu, J., and J. B. Kaper. 1992. Cloning and characterization of the eae geneof enterohaemorrhagic Escherichia coli O157:H7. Mol. Microbiol. 6:411–417.

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Expression of Intimin g from Enterohemorrhagic Escherichia ... · The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule intimin include the recep-tor-binding